Micro-RNAS of the MIR-15 family modulate cardiomyocyte survival and cardiac repair

ABSTRACT

A family of microRNAs, called the miR-15 family, which includes miR-195, are shown to be up-regulated during pathological cardiac remodeling and repress the expression of mRNAs required for cell proliferation and survival, with consequent loss of cardiomyocytes. Strategies to block expression of the miR-15 family in the heart as a treatment for diverse cardiac disease are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application of InternationalApplication No. PCT/US2008/083020, filed Nov. 10, 2008, which claims thebenefit of U.S. Provisional Application No. 60/986,798, filed Nov. 9,2007, which is herein incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with grant support under grant no. HL53351-06from the National Institutes of Health. The government has certainrights in the invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:MIRG_(—)002_(—)01US_SeqList_ST25.txt, date recorded: Aug. 23, 2010, filesize 4 kilobytes).

FIELD OF THE INVENTION

The present invention relates generally to the fields of developmentalbiology and molecular biology. More particularly, it concerns generegulation and cellular physiology in cardiomyocytes. Specifically, theinvention relates to a family of miRNAs, designated as the miR-15family, that regulate cardiomyocyte survival and cardiac repair.Inhibition of these miRNAs provides for reduced apoptosis in cardiaccells and thus inhibits cardiac hypertrophy and heart failure.

BACKGROUND OF THE INVENTION

Heart disease and its manifestations, including coronary artery disease,myocardial infarction, congestive heart failure and cardiac hypertrophy,clearly present a major health risk in the United States today. The costto diagnose, treat and support patients suffering from these diseases iswell into the billions of dollars. Two particularly severemanifestations of heart disease are myocardial infarction and cardiachypertrophy. With respect to myocardial infarction, typically an acutethrombocytic coronary occlusion occurs in a coronary artery as a resultof atherosclerosis and causes myocardial cell death. Becausecardiomyocytes, the heart muscle cells, are terminally differentiatedand generally incapable of cell division, they are generally replaced byscar tissue when they die during the course of an acute myocardialinfarction. Scar tissue is not contractile, fails to contribute tocardiac function, and often plays a detrimental role in heart functionby expanding during cardiac contraction, or by increasing the size andeffective radius of the ventricle, for example, becoming hypertrophic.

With respect to cardiac hypertrophy, one theory regards this as adisease that resembles aberrant development and, as such, raises thequestion of whether developmental signals in the heart can contribute tohypertrophic disease. Cardiac hypertrophy is an adaptive response of theheart to virtually all forms of cardiac disease, including those arisingfrom hypertension, mechanical load, myocardial infarction, cardiacarrhythmias, endocrine disorders, and genetic mutations in cardiaccontractile protein genes. While the hypertrophic response is initiallya compensatory mechanism that augments cardiac output, sustainedhypertrophy can lead to dilated cardiomyopathy (DCM), heart failure, andsudden death. In the United States, approximately half a millionindividuals are diagnosed with heart failure each year, with a mortalityrate approaching 50%. The causes and effects of cardiac hypertrophy havebeen extensively documented, but the underlying molecular mechanismshave not been elucidated. Understanding these mechanisms is a majorconcern in the prevention and treatment of cardiac disease and will becrucial as a therapeutic modality in designing new drugs thatspecifically target cardiac hypertrophy and cardiac heart failure.

Treatment with pharmacological agents still represents the primarymechanism for reducing or eliminating the manifestations of heartfailure. Diuretics constitute the first line of treatment formild-to-moderate heart failure. If diuretics are ineffective,vasodilatory agents, such as angiotensin converting enzyme (ACE)inhibitors (e.g., enalopril and lisinopril) or inotropic agent therapy(i.e., a drug that improves cardiac output by increasing the force ofmyocardial muscle contraction) may be used. Unfortunately, many of thesestandard therapies have numerous adverse effects and are contraindicatedin some patients. Thus, the currently used pharmacological agents havesevere shortcomings in particular patient populations. The availabilityof new, safe and effective agents would undoubtedly benefit patients whoeither cannot use the pharmacological modalities presently available, orwho do not receive adequate relief from those modalities.

The adult heart is a dynamic organ capable of significant remodeling andhypertrophic growth as a means of adapting function to altered workloadsor injury. Hemodynamic stress or neuroendocrine signaling associatedwith myocardial infarction, hypertension, aortic stenosis, and valvulardysfunction evoke a pathologic remodeling response through theactivation of intracellular signaling pathways and transcriptionalmediators in cardiac myocytes. Activation of these molecular pathwaysenhances cardiomyocyte size and protein synthesis, induces the assemblyof sarcomeres, and causes reexpression of fetal cardiac genes. Althoughaspects of the hypertrophic response after acute and chronic stress mayinitially augment cardiac output, prolonged hypertrophy is a majorpredictor of heart failure and sudden death. There have been majoradvances in the identification of genes and signaling pathways involvedin this disease process, but the overall complexity of hypertrophicremodeling suggests that additional regulatory mechanisms remain to beidentified.

MicroRNAs (miRNAs or miRs) have recently been implicated in a number ofbiological processes including regulation of developmental timing,apoptosis, fat metabolism, and hematopoietic cell differentiation amongothers. mRNAs are small, non-protein coding RNAs of about 18 to about 25nucleotides in length that regulate gene expression in asequence-specific manner mRNAs act as repressors of target mRNAs bypromoting their degradation, when their sequences are perfectlycomplementary, or by inhibiting translation, when their sequencescontain mismatches.

mRNAs are transcribed by RNA polymerase II (pol II) or RNA polymeraseIII (pol III; see Qi et al. (2006) Cellular & Molecular Immunology Vol.3:411-419) and arise from initial transcripts, termed primary miRNAtranscripts (pri-miRNAs), that are generally several thousand bases longand are derived from individual miRNA genes, from introns of proteincoding genes, or from poly-cistronic transcripts that often encodemultiple, closely related miRNAs. See review of Carrington et al.(2003). Pri-miRNAs are processed in the nucleus by the RNase Drosha intoabout 70- to about 100-nucleotide hairpin-shaped precursors(pre-miRNAs). Following transport to the cytoplasm, the hairpinpre-miRNA is further processed by Dicer to produce a double-strandedmiRNA (Lee et al., 1993). The mature miRNA strand is then incorporatedinto the RNA-induced silencing complex (RISC), where it associates withits target mRNAs by base-pair complementarity. In the relatively rarecases in which a miRNA base pairs perfectly with an mRNA target, itpromotes mRNA degradation. More commonly, miRNAs form imperfectheteroduplexes with target mRNAs, affecting either mRNA stability orinhibiting mRNA translation.

The 5′ portion of a miRNA spanning bases 2-8, termed the ‘seed’ region,is especially important for target recognition (Krenz and Robbins, 2004;Kiriazis and Kranias, 2000). The sequence of the seed, together withphylogenetic conservation of the target sequence, forms the basis formany current target prediction models. Although increasinglysophisticated computational approaches to predict miRNAs and theirtargets are becoming available, target prediction remains a majorchallenge and requires experimental validation. Ascribing the functionsof miRNAs to the regulation of specific mRNA targets is furthercomplicated by the ability of individual miRNAs to base pair withhundreds of potential high and low affinity mRNA targets and by thetargeting of multiple miRNAs to individual mRNAs.

The high sequence conservation of many miRNAs across metazoan speciessuggests strong evolutionary pressure and participation in essentialbiologic processes (Reinhart et al., 2000; Stark et al., 2005). Indeed,miRNAs have been shown to play fundamental roles in diverse biologicaland pathological processes, including cell proliferation,differentiation, apoptosis, and carcinogenesis in species ranging fromCaenorhabditis elegans and Drosophila melanogaster to humans. However,there remains limited information on the role that miRNAs play incardiogenesis and molecular events that can contribute to heart disease.

SUMMARY OF THE INVENTION

The present invention provides a method of treating pathologic cardiachypertrophy, heart failure, or myocardial infarction in a subject inneed thereof. In one embodiment, the method comprises identifying asubject having cardiac hypertrophy, heart failure, or myocardialinfarction; and inhibiting expression or activity of one or more miR-15family members in heart cells of said subject. In another embodiment,the method further comprises administering to the subject a secondtherapy. The second therapy may be, for example, a beta blocker, anionotrope, a diuretic, ACE inhibitor, AII antagonist, BNP, aCa++-blocker, and ERA, or an HDAC inhibitor.

In some embodiments of the invention, inhibiting the expression oractivity of one or more miR-15 family members comprises administering anantagomir of one or more miR-15 family members. In one embodiment, theexpression or activity of one or more miR-15 family members is inhibitedby administering an antisense oligonucleotide that targets the maturesequence of a miR-15 family member. In yet another embodiment,expression or activity of one or more miR-15 family members is inhibitedby administering an inhibitory RNA molecule, wherein the inhibitory RNAmolecule comprises a double stranded region that is at least partiallyidentical and complementary to a mature sequence of a miR-15 familymember. The inhibitory RNA molecule may be a ribozyme, siRNA or shRNAmolecule. In still another embodiment, expression or activity of one ormore miR-15 family members is inhibited by administering a nucleic acidcomprising one or more miR-15 binding sites. A miR-15 binding site maycomprise a sequence that is complementary to a seed sequence of miR-15.The one or more miR-15 family members may be miR-15a, miR-15b, miR-16-1,miR-16-2, miR-195, miR-424 and miR-497.

The present invention also provides a method of preventing pathologichypertrophy or heart failure in a subject in need thereof. In oneembodiment, the method comprises identifying a subject at risk ofdeveloping pathologic cardiac hypertrophy or heart failure; andinhibiting expression or activity of one or more miR-15 family membersin heart cells of said subject. In one embodiment, inhibiting comprisesdelivering to the heart cells an inhibitor of one or more miR-15 familymembers. In another embodiment, the subject at risk may exhibit one ormore risk factors selected from the group consisting of uncontrolledhypertension, uncorrected valvular disease, chronic angina, recentmyocardial infarction, congenital predisposition to heart disease, andpathological hypertrophy.

Antagomirs, antisense oligonucleotides, inhibitory RNA molecules,nucleic acids comprising miR-15 binding sites or other modulators of theexpression or activity of one or more miR-15 family members may beadministered by any method known to those in the art suitable fordelivery to the targeted organ, tissue, or cell type. For example, incertain embodiments of the invention, the modulator of one or moremiR-15 family members may be administered by parenteral administration,such as intravenous injection, intraarterial injection, intrapericardialinjection, or subcutaneous injection, or by direct injection into thetissue (e.g., cardiac tissue). In some embodiments, the modulator of oneor more miR-15 family members may be administered by oral, transdermal,intraperitoneal, subcutaneous, sustained release, controlled release,delayed release, suppository, or sublingual routes of administration. Inother embodiments, the modulator of one or more miR-15 family membersmay be administered by a catheter system.

The present invention also encompasses a transgenic, non-human mammal,the cells of which fail to express a functional form of one or moremiR-15 family members (e.g. miR-15a, miR-15b, miR-16-1, miR-16-2,miR-195, miR-424 and miR-497). In another embodiment, the presentinvention includes a transgenic, non-human mammal, the cells of whichcomprise a coding region of a miR-15 family member under the control ofa heterologous promoter active in the cells of said non-human mammal. Insome embodiments, the mammal is a mouse.

The present invention provides a method for identifying a modulator of amiR-15 family member comprising (a) contacting a cell with a candidatecompound; (b) assessing activity or expression of an miR-15 familymember; and (c) comparing the activity or expression in step (b) withthe activity or expression in the absence of the candidate compound,wherein a difference between the measured activities or expressionindicates that the candidate compound is a modulator of said miR-15family member. The cell may be contacted with the candidate compound invitro or in vivo. The candidate compound may be a protein, a peptide, apolypeptide, a polynucleotide, an oligonucleotide, or small molecule.The modulator of a miR-15 family member may be an agonist or inhibitorof the miR-15 family member. The modulator of a miR-15 family member maybe an agonist or inhibitor of an upstream regulator of the miR-15 familymember.

The present invention also provides a pharmaceutical compositioncomprising an inhibitor of one or more miR-15 family members. In oneembodiment, the composition is formulated for injection. In anotherembodiment, the pharmaceutical composition is combined with a kit foradministration, such as parenteral or catheter administration.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. miRNA expression during cardiac hypertrophy. A. H&E stainedsections of representative hearts from mice following sham and thoracicaortic banding (TAB) for 21 days and from wild-type (WT) and activatedcalcineurin transgenic (CnA Tg) mice. Scale bar equals 2 mm. B. Numbersof miRNAs that were regulated in response to CnA or TAB are indicated.Although some changes were unique for either TAB or CnA-inducedhypertrophy, most miRNAs that were induced or repressed overlapped forthe different hypertrophic stimuli. C. Northern blot analysis ofparticular miRNAs in WT and CnA Tg hearts. U6 RNA was detected as aloading control.

FIG. 2. miRNA expression in human heart failure. Northern blot analysisof miRNAs in 4 normal and 6 failing human hearts. The averagefold-change of each miRNA in the failing samples is shown at the right.

FIG. 3. Cardiac specific over-expression of miRNA 195 is sufficient todrive cardiomyopathy. H&E stained sections of hearts from wild-type (WT)and two different lines of miR-195 transgenic (Tg) animals. miRNA-195 Tgline 3 animals died two weeks after birth due to cardiac dilation.Northern blot analysis on hearts from WT and miR-195 transgenic lines 1and 3 confirming a 26.5-fold and 29.2-fold cardiac specific miRNAover-expression, respectively.

FIG. 4. Overexpression of miR-195 induces cardiac dysfunction due tocardiac growth. A. Echocardiographic analyses indicate miR-195transgenic (Tg) mice show left ventricular (LV) dilation and wallthinning, resulting in a decreased fractional shortening compared towild-type (WT) littermates. B. Heart weight to body weight ratioincreases in response to cardiac specific overexpression of miR-195. C.Realtime PCR analysis shows an upregulation of hypertrophic genes inmiR-195 Tg animals compared to WT animals (n=3). *P<0.05 compared towild-type.

FIG. 5. Cardiac remodeling specific for miR-195. Over-expression ofmiR-195 induces cardiac growth at 2 weeks of age, which within 6 weeksprogresses to a dilated phenotype. Cardiac over-expression of miR-214has no phenotypic effect, indicating the specific effect of miR-195 oncardiac pathology. Scale bar equals 2 mm.

FIG. 6. MiR-195 is part of the miR-15 family that targets pro-survivalproteins. A. MiR-195 is part of the miR-15 family that consists of fivedifferent miRs: miR-15, miR-16, miR-195, miR-424, and miR-497. Four ofthe miR-15 family members are expressed as three clusters of two miRNAs.B. MiR-15 family members target proteins involved in proliferation,survival and anti-apoptosis. Thus, up-regulation of miR-195 results indown-regulation of these mRNAs and cell death.

FIG. 7. MiR-195 target sequence in the 3′ UTR of FGF2 mRNA.

FIG. 8. Enhanced expression of miR-15 family members in samples fromfailing human hearts. A. Left panel shows RNA blots from normal humanhearts and right panel shows RNA blots from failing human hearts. B. Theconserved seed region among miR-15 family members allows for anadditional approach to knockdown the whole miR-15 family. This approachentails overexpression of multiple miR-15 binding sites under thecontrol of a cardiac specific promoter, such as the alpha myosin heavychain promoter (aMHC). Each of the miR-15 binding sites contains asequence complementary to the sequence of the conserved seed region andallows for the scavenging of all family members, thereby preventing themfrom binding to their endogenous targets.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is based, in part, on the discovery that membersof the miR-15 family of microRNAs, such as miR-195, are upregulated inmyocardial tissue from human failing hearts as well as in animal modelsof pathologic cardiac hypertrophy. Cardiac overexpression of miR-195 issufficient to induce cardiac hypertrophy and can lead to a dilatedphenotype. Accordingly, the present invention provides methods oftreating or preventing various forms of heart disease in a subject byinhibiting the expression or activity of one or more members of themiR-15 family in heart cells of the subject.

The miR-15 family is a small family of microRNAs that includes miR-195,miR-16-1, miR-15a, miR-15b, miR-16-2, miR-424, and miR-497. Four of themiR-15 family members are expressed as three clustered transcripts (FIG.6A). MiR-195 and miR-497 are expressed as a cluster from the intron of agene located on chromosome 17. There are two copies of miR-16 located ondifferent chromosomes. One copy (miR-16-1) is expressed as a clusterwith miR-15a from an intron of a gene encoded on chromosome 13. Thesecond copy (miR-16-2) is expressed as a cluster with 15b from theintron of the SMC4 gene located on chromosome 3. MiR-424 is expressedfrom the X chromosome. The pre-miRNA sequences (e.g. stem loopsequences) for each of the miR-15 family members are listed below:

Human pre-miR-195 (SEQ ID NO: 1)AGCUUCCCUG GCUCUAGCAG CACAGAAAUA UUGGCACAGGGAAGCGAGUC UGCCAAUAUU GGCUGUGCUG CUCCAGGCAG GGUGGUG Human pre-miR-497(SEQ ID NO: 2) CCACCCCGGU CCUGCUCCCG CCCCAGCAGC ACACUGUGGUUUGUACGGCA CUGUGGCCAC GUCCAAACCA CACUGUGGUGUUAGAGCGAG GGUGGGGGAG GCACCGCCGA GG Human pre-miR-16-1 (SEQ ID NO: 3)GUCAGCAGUG CCUUAGCAGC ACGUAAAUAU UGGCGUUAAGAUUCUAAAAU UAUCUCCAGU AUUAACUGUG CUGCUGAAGU AAGGUUGAC Human pre-miR-16-2(SEQ ID NO: 4) GUUCCACUCU AGCAGCACGU AAAUAUUGGC GUAGUGAAAUAUAUAUUAAA CACCAAUAUU ACUGUGCUGC UUUAGUGUGA C Human pre-miR-15a(SEQ ID NO: 5) CCUUGGAGUA AAGUAGCAGC ACAUAAUGGU UUGUGGAUUUUGAAAAGGUG CAGGCCAUAU UGUGCUGCCU CAAAAAUACA AGG Human pre-miR-15b(SEQ ID NO: 6) UUGAGGCCUU AAAGUACUGU AGCAGCACAU CAUGGUUUACAUGCUACAGU CAAGAUGCGA AUCAUUAUUU GCUGCUCUAG AAAUUUAAGG AAAUUCAUHuman pre-miR-424 (SEQ ID NO: 19)CGAGGGGAUA CAGCAGCAAU UCAUGUUUUG AAGUGUUCUAAAUGGUUCAA AACGUGAGGC GCUGCUAUAC CCCCUCGUGG GGAAGGUAGA AGGUGGGG

Each of the pre-miRNA sequences for each miR-15 family member isprocessed into a mature sequence and a star sequence. The star sequenceis processed from the other strand of the stem loop structure. Themature and star sequences for each of the miR-15 family members is givenbelow:

Human mature miR-195 UAGCAGCACAGAAAUAUUGGC (SEQ ID NO: 7) Human miR-195*CCAAUAUUGGCUGUGCUGCUCC (SEQ ID NO: 8) Human mature miR-497CAGCAGCACACUGUGGUUUGU (SEQ ID NO: 9) Human miR-497*CAAACCACACUGUGGUGUUAGA (SEQ ID NO: 10) Human mature miR-16-1/m1R-16-2UAGCAGCACGUAAAUAUUGGCG (SEQ ID NO: 11) Human miR-16-1*CCAGUAUUAACUGUGCUGCUGA (SEQ ID NO: 12) Human miR-16-2*CCAAUAUUACUGUGCUGCUUUA (SEQ ID NO: 13) Human mature miR-15aUAGCAGCACAUAAUGGUUUGUG (SEQ ID NO: 14) Human miR-15a*CAGGCCAUAUUGUGCUGCCUCA (SEQ ID NO: 15) Human mature miR-15bUAGCAGCACAUCAUGGUUUACA (SEQ ID NO: 16) Human miR-15b*CGAAUCAUUAUUUGCUGCUCUA (SEQ ID NO: 17) Human mature miR-424CAGCAGCAAUUCAUGUUUUGAA (SEQ ID NO: 20) Human miR-424*CAAAACGUGAGGCGCUGCUAU (SEQ ID NO: 21)

Although the seed region (e.g. bases spanning 2 to 8 nucleotides ofmature miRNA sequence) for all family members is highly conserved(AGCAGCAC; SEQ ID NO: 18), the 3′ end of the mature miRNA differs amongthe different family members (FIG. 6A).

The present invention provides a method of treating pathologic cardiachypertrophy, heart failure, or myocardial infarction in a subject inneed thereof by inhibiting expression or activity of one or more miR-15family members. In one embodiment, the method comprises identifying asubject having cardiac hypertrophy, heart failure, or myocardialinfarction; and inhibiting expression or activity of one or more miR-15family members in heart cells of said subject. “Heart cells” as usedherein include cardiomyocytes, cardiac fibroblasts, and cardiacendothelial cells. In another embodiment, the method comprisesadministering to the subject an inhibitor of one or more miR-15 familymembers. In still another embodiment, the method comprises identifying asubject at risk of developing pathologic cardiac hypertrophy or heartfailure and inhibiting expression or activity of one or more miR-15family members in heart cells of the subject. The subject at risk ofdeveloping pathologic cardiac hypertrophy or heart failure may exhibitone or more risk factors including, for example, uncontrolledhypertension, uncorrected valvular disease, chronic angina, recentmyocardial infarction, congenital predisposition to heart disease orpathological hypertrophy. In certain embodiments, the subject at riskmay be diagnosed as having a genetic predisposition to cardiachypertrophy. In some embodiments of the invention, the subject at riskmay have a familial history of cardiac hypertrophy.

In another embodiment, the present invention provides a method ofpreventing cardiac hypertrophy and dilated cardiomyopathy in a subjectin need thereof comprising inhibiting expression or activity of one ormore miR-15 family members in heart cells of the subject. In yet afurther embodiment, the present invention provides a method ofinhibiting progression of cardiac hypertrophy in a subject in needthereof comprising inhibiting expression or activity of one or moremiR-15 family members in heart cells of the subject. In furtherembodiments, the present invention provides a method of increasingexercise tolerance, reducing hospitalization, improving quality of life,decreasing morbidity, and/or decreasing mortality in a subject withheart failure or cardiac hypertrophy comprising inhibiting expression oractivity of one or more miR-15 family members in heart cells of thesubject.

Thus, the present invention provides methods for the treatment ofcardiac hypertrophy, heart failure, or myocardial infarction utilizinginhibitors of miR-15 family members, such as miR-195, miR-15a, miR-15b,miR-16-1, miR-16-2, miR-424, and miR-497. Preferably, administration ofan inhibitor of a miR-15 family member results in the improvement of oneor more symptoms of cardiac hypertrophy, heart failure, or myocardialinfarction in the subject, or in the delay in the transition fromcardiac hypertrophy to heart failure. The one or more improved symptomsmay be, for example, increased exercise capacity, increased cardiacejection volume, decreased left ventricular end diastolic pressure,decreased pulmonary capillary wedge pressure, increased cardiac output,increased cardiac index, lowered pulmonary artery pressures, decreasedleft ventricular end systolic and diastolic dimensions, decreased leftand right ventricular wall stress, decreased wall tension, increasedquality of life, and decreased disease related morbidity or mortality.In addition, use of inhibitors of miR-15 family members may preventcardiac hypertrophy and its associated symptoms from arising. In oneembodiment, administration of an inhibitor of one or more miR-15 familymembers to a subject suffering from myocardial infarction may reduceinfarct size by decreasing the loss of heart cells. In anotherembodiment, cardiac function is stabilized in a subject suffering frommyocardial infarction following administration of an inhibitor of one ormore miR-15 family members.

Inhibition of microRNA function may be achieved by administeringantisense oligonucleotides targeting a mature sequence of a miR-15family member. The antisense oligonucleotides may be ribonucleotides ordeoxyribonucleotides. Preferably, the antisense oligonucleotides have atleast one chemical modification. Antisense oligonucleotides may becomprised of one or more “locked nucleic acids”. “Locked nucleic acids”(LNAs) are modified ribonucleotides that contain an extra bridge betweenthe 2′ and 4′ carbons of the ribose sugar moiety resulting in a “locked”conformation that confers enhanced thermal stability to oligonucleotidescontaining the LNAs. Alternatively, the antisense oligonucleotides maycomprise peptide nucleic acids (PNAs), which contain a peptide-basedbackbone rather than a sugar-phosphate backbone. Other chemicalmodifications that the antisense oligonucleotides may contain include,but are not limited to, sugar modifications, such as 2′-O-alkyl (e.g.2′-O-methyl, 2′-O-methoxyethyl), 2′-fluoro, and 4′ thio modifications,and backbone modifications, such as one or more phosphorothioate,morpholino, or phosphonocarboxylate linkages (see, for example, U.S.Pat. Nos. 6,693,187 and 7,067,641, which are herein incorporated byreference in their entireties). In some embodiments, suitable antisenseoligonucleotides are 2′-O-methoxyethyl “gapmers” which contain2′-O-methoxyethyl-modified ribonucleotides on both 5′ and 3′ ends withat least ten deoxyribonucleotides in the center. These “gapmers” arecapable of triggering RNase H-dependent degradation mechanisms of RNAtargets. Other modifications of antisense oligonucleotides to enhancestability and improve efficacy, such as those described in U.S. Pat. No.6,838,283, which is herein incorporated by reference in its entirety,are known in the art and are suitable for use in the methods of theinvention. Preferable antisense oligonucleotides useful for inhibitingthe activity of microRNAs are about 19 to about 25 nucleotides inlength. Antisense oligonucleotides may comprise a sequence that is atleast partially complementary to a mature miRNA sequence, e.g. at leastabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to amature miRNA sequence. In some embodiments, the antisenseoligonucleotide may be substantially complementary to a mature miRNAsequence, that is at least about 95%, 96%, 97%, 98%, or 99%complementary to a target polynucleotide sequence. In one embodiment,the antisense oligonucleotide comprises a sequence that is 100%complementary to a mature miRNA sequence.

In some embodiments, the antisense oligonucleotides are antagomirs.“Antagomirs” are single-stranded, chemically-modified ribonucleotidesthat are at least partially complementary to the miRNA sequence.Antagomirs may comprise one or more modified nucleotides, such as2′-O-methyl-sugar modifications. In some embodiments, antagomirscomprise only modified nucleotides. Antagomirs may also comprise one ormore phosphorothioate linkages resulting in a partial or fullphosphorothioate backbone. To facilitate in vivo delivery and stability,the antagomir may be linked to a cholesterol or other moiety at its 3′end. Antagomirs suitable for inhibiting miRNAs may be about 15 to about50 nucleotides in length, more preferably about 18 to about 30nucleotides in length, and most preferably about 20 to about 25nucleotides in length. “Partially complementary” refers to a sequencethat is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%complementary to a target polynucleotide sequence. The antagomirs may beat least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%complementary to a mature miRNA sequence. In some embodiments, theantagomir may be substantially complementary to a mature miRNA sequence,that is at least about 95%, 96%, 97%, 98%, or 99% complementary to atarget polynucleotide sequence. In other embodiments, the antagomirs are100% complementary to the mature miRNA sequence.

Another approach for inhibiting the function of a miR-15 family memberis administering an inhibitory RNA molecule having a double strandedregion that is at least partially identical and partially complementaryto a mature sequence of the miR-15 family member. The inhibitory RNAmolecule may be a double-stranded, small interfering RNA (siRNA) or ashort hairpin RNA molecule (shRNA) comprising a stem-loop structure. Thedouble-stranded regions of the inhibitory RNA molecule may comprise asequence that is at least partially identical and partiallycomplementary, e.g. about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical and complementary, to the mature miRNA sequence. In someembodiments, the double-stranded regions of the inhibitory RNA comprisea sequence that is at least substantially identical and substantiallycomplementary to the mature miRNA sequence. “Substantially identical andsubstantially complementary” refers to a sequence that is at least about95%, 96%, 97%, 98%, or 99% identical and complementary to a targetpolynucleotide sequence. In other embodiments, the double-strandedregions of the inhibitory RNA molecule may contain 100% identity andcomplementarity to the target miRNA sequence.

The inhibitory nucleotide molecules described herein preferably target amature sequence of one or more miR-15 family members (e.g. SEQ ID NOs:7, 9, 11, 14, 16, and 20) or a star sequence of one or more miR-15family members (e.g. SEQ ID NOs: 8, 10, 12, 13, 15, 17, and 21). In someembodiments, inhibitors of miR-15 family members are antagomirscomprising a sequence that is perfectly complementary to a maturesequence of a miR-15 family member. In one embodiment, an inhibitor of amiR-15 family member is an antagomir having a sequence that is partiallyor perfectly complementary to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 20. In another embodiment,an inhibitor of a miR-15 family member is an antagomir having a sequencethat is partially or perfectly complementary to SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQID NO: 21.

In some embodiments, inhibitors of one or more miR-15 family members arechemically-modified antisense oligonucleotides. In one embodiment, aninhibitor of a miR-15 family member is a chemically-modified antisenseoligonucleotide comprising a sequence substantially complementary to SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, orSEQ ID NO: 20. In another embodiment, an inhibitor of a miR-15 familymember is a chemically-modified antisense oligonucleotide comprising asequence substantially complementary to SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO:21. As used herein “substantially complementary” refers to a sequencethat is at least about 95%, 96%, 97%, 98%, 99%, or 100% complementary toa target polynucleotide sequence (e.g. mature or precursor miRNAsequence).

Antisense oligonucleotides may comprise a sequence that is substantiallycomplementary to a precursor miRNA sequence (pre-miRNA) for one or moremiR-15 family members (e.g. pre-miR-195, pre-miR-497, pre-miR-424,pre-miR-15a, pre-miR-15b, pre-miR-16-1, or pre-miR-16-2). In someembodiments, the antisense oligonucleotide comprises a sequence that issubstantially complementary to a sequence located outside the stem-loopregion of the pre-miRNA sequence. In one embodiment, an inhibitor of amiR-15 family member is an antisense oligonucleotide having a sequencethat is substantially complementary to a pre-miRNA sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 19.

In other embodiments of the invention, inhibitors of one or more miR-15family members may be inhibitory RNA molecules, such as ribozymes,siRNAs, or shRNAs. In one embodiment, an inhibitor of a miR-15 familymember is an inhibitory RNA molecule comprising a double-strandedregion, wherein the double-stranded region comprises a sequence having100% identity and complementarity to a mature sequence of a miR-15family member (e.g. SEQ ID NOs: 7, 9, 11, 14, 16, and 20). In anotherembodiment, an inhibitor of a miR-15 family member is an inhibitory RNAmolecule comprising a double-stranded region, wherein thedouble-stranded region comprises a sequence having 100% identity andcomplementarity to a star sequence of a miR-15 family member (e.g. SEQID NOs: 8, 10, 12, 13, 15, 17, and 21). In some embodiments, inhibitorsof one or more miR-15 family members are inhibitory RNA molecules whichcomprise a double-stranded region, wherein said double-stranded regioncomprises a sequence of at least about 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity and complementarity to a mature sequence ofone or more miR-15 family members.

Multiple members of the miR-15 family (e.g. miR-15a, miR-15b, miR-16-1,miR-16-2, miR-195, miR-424, and miR-497) may be inhibited simultaneouslyby administering multiple inhibitors, wherein each inhibitor targets aseparate miR-15 family member. For example, in some embodiments, atleast two members of the miR-15 family are inhibited by administeringtwo separate inhibitors. In other embodiments, at least three members ofthe miR-15 family are inhibited by administering three separateinhibitors. In still other embodiments, at least four members of themiR-15 family are inhibited by administering four separate inhibitors.In further embodiments, at least five members of the miR-15 family areinhibited by administering five separate inhibitors. In one embodiment,all six members of the miR-15 family are inhibited by administering sixseparate inhibitors.

In another embodiment, an inhibitor of miR-15 family function is anucleic acid comprising one or more miR-15 binding sites. The seedregion (AGCAGCAC; SEQ ID NO: 18) of all the miR-15 family members ishighly conserved. Therefore, a nucleic acid comprising a binding sitehaving substantial complementarity to the miR-15 seed sequence wouldbind all members of the miR-15 family. This approach has been analogizedto the use of a sponge to “soak up” the effective miR-15 family members,thereby reducing the overall pool of miRNAs that could impact a giventarget sequence. The term “miR-15 binding site” as used herein refers toa nucleotide sequence that is capable of binding a mature sequence ofmiR-15a, miR-15b, miR-16-1, miR-16-2, miR-195, miR-424, miR-497, orcombinations thereof. Preferably, a miR-15 binding site comprises asequence that is substantially complementary to the miR-15 seedsequence. The seed sequence or seed region refers to nucleotides 2-8 ofthe 5′ portion of the mature miRNA sequence. In one embodiment, themiR-15 binding site comprises the a sequence that is substantiallycomplementary to of SEQ ID NO: 18. The inhibitory nucleic acidcomprising one or more miR-15 binding sites may be from about 20 toabout 500 nucleotides in length, about 25 to about 400 nucleotides inlength, about 30 to about 300 nucleotides in length, about 40 to about200 nucleotides in length, or about 50 to about 100 nucleotides inlength. For example, the nucleic acid may be 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300,350, 400, 500 nucleotides in length. The nucleic acid may contain 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 miR-15 binding sites. Themultiple miR-15 binding sites may be adjacent or may be separated byspacers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

In another embodiment, an expression vector may be used to deliver aninhibitor of one or more miR-15 family members to a cell or subject. A“vector” is a composition of matter which can be used to deliver anucleic acid of interest to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “vector” includes an autonomouslyreplicating plasmid or a virus. Examples of viral vectors include, butare not limited to, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, and the like. An expression construct can bereplicated in a living cell, or it can be made synthetically. Forpurposes of this application, the terms “expression construct,”“expression vector,” and “vector,” are used interchangeably todemonstrate the application of the invention in a general, illustrativesense, and are not intended to limit the invention.

In one embodiment, an expression vector for expressing an inhibitor ofone or more miR-15 family members comprises a promoter operably linkedto a polynucleotide encoding an antisense oligonucleotide, wherein thesequence of the expressed antisense oligonucleotide is partially orperfectly complementary to a mature sequence of one or more miR-15family members. The phrase “operably linked” or “under transcriptionalcontrol” as used herein means that the promoter is in the correctlocation and orientation in relation to a polynucleotide to control theinitiation of transcription by RNA polymerase and expression of thepolynucleotide. In another embodiment, an expression vector forexpressing an inhibitor of one or more miR-15 family members comprisesone or more promoters operably linked to a polynucleotide encoding ashRNA or siRNA, wherein the expressed shRNA or siRNA comprises a doublestranded region that is identical and complementary or partiallyidentical and partially complementary to a mature sequence of one ormore miR-15 family members. “Partially identical and partiallycomplementary” refers to a sequence that is at least about 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical and complementary to atarget polynucleotide sequence.

In certain embodiments, the nucleic acid encoding a polynucleotide ofinterest is under transcriptional control of a promoter. A “promoter”refers to a DNA sequence recognized by the synthetic machinery of thecell, or introduced synthetic machinery, required to initiate thespecific transcription of a gene. The term promoter will be used here torefer to a group of transcriptional control modules that are clusteredaround the initiation site for RNA polymerase I, II, or III.

In some embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter, and glyceraldehyde-3-phosphatedehydrogenase promoter can be used to obtain high-level expression ofthe coding sequence of interest. The use of other viral or mammaliancellular or bacterial phage promoters which are well-known in the art toachieve expression of a coding sequence of interest is contemplated aswell, provided that the levels of expression are sufficient for a givenpurpose.

By employing a promoter with well-known properties, the level andpattern of expression of the polynucleotide of interest followingtransfection or transformation can be optimized. Further, selection of apromoter that is regulated in response to specific physiologic signalscan permit inducible expression of the gene product. Tables 1 and 2 listseveral regulatory elements that may be employed, in the context of thepresent invention, to regulate the expression of the polynucleotide ofinterest (e.g. inhibitor of miR-15 family members). This list is notintended to be exhaustive of all the possible elements involved in thepromotion of gene expression but, merely, to be exemplary thereof.

Below is a list of viral promoters, cellular promoters/enhancers andinducible promoters/enhancers that could be used in combination with thepolynucleotide of interest in an expression construct (Table 1 and Table2). Additionally, any promoter/enhancer combination (as per theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

TABLE 1 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOrnitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culottaet al., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987aAlbumin Pinkert et al., 1987; Tronche et al., 1989, 1990 α-FetoproteinGodbout et al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al.,1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al.,1987; Quinn et al., 1989

TABLE 2 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Heavy metals Haslinger et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouseGlucocorticoids Huang et al., 1981; Lee mammary tumor virus) et al.,1981; Majors et al., 1983; Chandler et al., 1983; Ponta et al., 1985;Sakai et al., 1988 β-Interferon poly(rI)x Tavernier et al., 1983poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 Collagenase PhorbolEster (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel etal., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX GeneInterferon, Hug et al., 1988 Newcastle Disease Virus GRP78 Gene A23187Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989 VimentinSerum Rittling et al., 1989 MHC Class I Gene Interferon Blanar et al.,1989 H-2κb HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a, Antigen1990b Proliferin Phorbol Ester-TPA Mordacq et al., 1989 Tumor NecrosisFactor PMA Hensel et al., 1989 Thyroid Stimulating Thyroid HormoneChatterjee et al., 1989 Hormone α Gene

Of particular interest are muscle specific promoters, and moreparticularly, cardiac specific promoters. These include the myosin lightchain-2 promoter (Franz et al., 1994; Kelly et al., 1995), the alphaactin promoter (Moss et al., 1996), the troponin 1 promoter (Bhavsar etal., 1996); the Na⁺/Ca²⁺ exchanger promoter (Barnes et al., 1997), thedystrophin promoter (Kimura et al., 1997), the alpha7 integrin promoter(Ziober and Kramer, 1996), the brain natriuretic peptide promoter(LaPointe et al., 1996) and the alpha B-crystallin/small heat shockprotein promoter (Gopal-Srivastava, 1995), alpha myosin heavy chainpromoter (Yamauchi-Takihara et al., 1989) and the ANF promoter (LaPointeet al., 1988).

A polyadenylation signal may be included to effect properpolyadenylation of the gene transcript where desired. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and any such sequence may be employed such ashuman growth hormone and SV40 polyadenylation signals. Also contemplatedas an element of the expression cassette is a terminator. These elementscan serve to enhance message levels and to minimize read through fromthe cassette into other sequences.

In certain embodiments of the invention, the cells containing nucleicacid constructs of the present invention, a cell may be identified invitro or in vivo by including a marker in the expression construct. Suchmarkers would confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Immunologic markers also can be employed. Theselectable marker employed is not believed to be important, so long asit is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

There are a number of ways in which expression vectors may be introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide (or other inhibitory polynucleotide) that has been clonedtherein. The expression vector comprises a genetically engineered formof adenovirus. Knowledge of the genetic organization of adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

The typical vector according to the present invention is replicationdefective and will not have an adenovirus E1 region. Thus, it will bemost convenient to introduce the polynucleotide encoding the gene ofinterest at the position from which the E1-coding sequences have beenremoved. However, the position of insertion of the construct within theadenovirus sequences is not critical to the invention. Thepolynucleotide encoding the gene of interest may also be inserted inlieu of the deleted E3 region in E3 replacement vectors, as described byKarlsson et al. (1986), or in the E4 region where a helper cell line orhelper virus complements the E4 defect.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

Retroviral vectors are also suitable for expressing inhibitors of miR-15family members in cells. The retroviruses are a group of single-strandedRNA viruses characterized by an ability to convert their RNA todouble-stranded DNA in infected cells by a process ofreverse-transcription (Coffin, 1990). The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a polynucleotide of interestmay also be transferred in a similar manner in vivo and express the geneproduct.

In still another embodiment of the invention for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular polynucleotideof interest may be delivered via this method and still be incorporatedby the present invention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a particular gene alsomay be specifically delivered into a cell type (e.g. cardiac cell) byany number of receptor-ligand systems with or without liposomes. Forexample, epidermal growth factor (EGF) may be used as the receptor formediated delivery of a nucleic acid into cells that exhibit upregulationof EGF receptor. Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

In a particular example, the polynucleotide may be administered incombination with a cationic lipid. Examples of cationic lipids include,but are not limited to, lipofectin, DOTMA, DOPE, and DOTAP. Thepublication of WO0071096, which is specifically incorporated byreference, describes different formulations, such as a DOTAP:cholesterolor cholesterol derivative formulation that can effectively be used forgene therapy. Other disclosures also discuss different lipid orliposomal formulations including nanoparticles and methods ofadministration; these include, but are not limited to, U.S. PatentPublication 20030203865, 20020150626, 20030032615, and 20040048787,which are specifically incorporated by reference to the extent theydisclose formulations and other related aspects of administration anddelivery of nucleic acids. Methods used for forming particles are alsodisclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835,5,972,901, 6,200,801, and 5,972,900, which are incorporated by referencefor those aspects.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

The present invention also includes methods for scavenging or clearinginhibitors of miR-15 family members following treatment. The method maycomprise overexpressing hybridization sites for inhibitors of the miR-15family members in cardiac tissue. In one embodiment, the methodcomprises overexpression of hybridization sites for inhibitors of themiR-15 family members in cardiac muscle using a heart muscle specificpromoter (e.g. α-MHC). In another embodiment, the hybridization site maycomprise a sequence of a seed region from a miR-15 family member. Inanother embodiment, the hybridization site may comprise the sequence ofSEQ ID NO: 18. In some embodiments, the hybridization site may contain asequence that is complementary to a sequence from the 3′UTR of one ormore targets of a miR-15 family member, such as FGF2, TGFb-inducedfactor 2, BCL9I, BCL2L, CDC25A, cyclin E1, cyclin D1, or cyclin D2.

In another embodiment of the invention, an inhibitor of one or moremiR-15 family members is administered to the subject in combination withother therapeutic modalities. Current medical management of cardiachypertrophy in the setting of a cardiovascular disorder includes the useof at least two types of drugs: inhibitors of the renin-angiotensinsystem and β-adrenergic blocking agents (Bristow, 1999). Therapeuticagents to treat pathologic hypertrophy in the setting of heart failureinclude angiotensin II converting enzyme (ACE) inhibitors andβ-adrenergic receptor blocking agents (Eichhorn and Bristow, 1996).Other pharmaceutical agents that have been disclosed for treatment ofcardiac hypertrophy include angiotensin II receptor antagonists (U.S.Pat. No. 5,604,251) and neuropeptide U antagonists (WO 98/33791).

Non-pharmacological treatment is primarily used as an adjunct topharmacological treatment. One means of non-pharmacological treatmentinvolves reducing the sodium in the diet. In addition,non-pharmacological treatment also entails the elimination of certainprecipitating drugs, including negative inotropic agents (e.g., certaincalcium channel blockers and antiarrhythmic drugs like disopyramide),cardiotoxins (e.g., amphetamines), and plasma volume expanders (e.g.,nonsteroidal anti-inflammatory agents and glucocorticoids).

Thus, in addition to the therapies described above, one may also provideto the subject more “standard” pharmaceutical cardiac therapies with theinhibitor of one or more miR-15 family members. Examples of othertherapies include, without limitation, so-called “beta blockers,”anti-hypertensives, cardiotonics, anti-thrombotics, vasodilators,hormone antagonists, iontropes, diuretics, endothelin receptorantagonists, calcium channel blockers, phosphodiesterase inhibitors, ACEinhibitors, angiotensin type 2 antagonists and cytokineblockers/inhibitors, and HDAC inhibitors. The combination therapy alsomay involve inhibiting the expression or activity of additional miRNAsinvolved in cardiac remodeling such as miR-499, miR-208, miR-208b andmiR-21. Combination therapy may also include overexpression ofparticular microRNAs, such as miR-29.

Combinations may be achieved by contacting cardiac cells with a singlecomposition or pharmacological formulation that includes an inhibitor ofone or more miR-15 family members and a standard pharmaceutical agent,or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes theinhibitor of a miR-15 family member and the other includes the standardpharmaceutical agent. Alternatively, the therapy using an inhibitor of amiR-15 family member may precede or follow administration of the otheragent(s) by intervals ranging from minutes to weeks. In embodimentswhere the standard pharmaceutical agent and the inhibitor of a miR-15family member are applied separately to the cell, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the pharmaceutical agent and inhibitor of amiR-15 family member would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone would typically contact the cell with both modalities within about12-24 hours of each other and, more preferably, within about 6-12 hoursof each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of either aninhibitor of a miR-15 family member, or the other pharmaceutical agentwill be desired. In this regard, various combinations may be employed.By way of illustration, where the inhibitor of a miR-15 family member is“A” and the other pharmaceutical agent is “B,” the followingpermutations based on 3 and 4 total administrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are likewise contemplated.

Treatment regimens would vary depending on the clinical situation.However, long-term maintenance would appear to be appropriate in mostcircumstances. It also may be desirable to treat hypertrophy withinhibitors of miR-15 family members intermittently, such as within abrief window during disease progression.

Pharmacological therapeutic agents and methods of administration,dosages, etc., are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Klaassen's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, ablood coagulant, an antiarrhythmic agent, an antihypertensive agent, avasopressor, a treatment agent for congestive heart failure, anantianginal agent, an antibacterial agent or a combination thereof.

In addition, it should be noted that any of the following may be used todevelop new sets of cardiac therapy target genes as β-blockers were usedin the present examples (see below). While it is expected that many ofthese genes may overlap, new gene targets likely can be developed.

In certain embodiments, administration of an agent that lowers theconcentration of one of more blood lipids and/or lipoproteins, knownherein as an “antihyperlipoproteinemic,” may be combined with acardiovascular therapy according to the present invention, particularlyin treatment of athersclerosis and thickenings or blockages of vasculartissues. In certain embodiments, an antihyperlipoproteinemic agent maycomprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acidsequesterant, a HMG CoA reductase inhibitor, a nicotinic acidderivative, a thyroid hormone or thyroid hormone analog, a miscellaneousagent or a combination thereof. Non-limiting examples ofaryloxyalkanoic/fibric acid derivatives include beclobrate, enzafibrate,binifibrate, ciprofibrate, clinofibrate, clofibrate (atromide-S),clofibric acid, etofibrate, fenofibrate, gemfibrozil (lobid),nicofibrate, pirifibrate, ronifibrate, simfibrate and theofibrate.Non-limiting examples of resins/bile acid sequesterants includecholestyramine (cholybar, questran), colestipol (colestid) andpolidexide. Non-limiting examples of HMG CoA reductase inhibitorsinclude lovastatin (mevacor), pravastatin (pravochol) or simvastatin(zocor). Non-limiting examples of nicotinic acid derivatives includenicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacicacid. Non-limiting examples of thyroid hormones and analogs thereofinclude etoroxate, thyropropic acid and thyroxine.

Non-limiting examples of miscellaneous antihyperlipoproteinemics includeacifran, azacosterol, benfluorex, β-benzalbutyramide, carnitine,chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium,5,8,11,14,17-eicosapentaenoic acid, eritadenine, furazabol, meglutol,melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine,pentaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol(lorelco), β-sitosterol, sultosilic acid-piperazine salt, tiadenol,triparanol and xenbucin. A non-limiting example of anantiarteriosclerotic includes pyridinol carbamate.

In certain embodiments, administration of an agent that aids in theremoval or prevention of blood clots may be combined with administrationof a modulator, particularly in treatment of athersclerosis andvasculature (e.g., arterial) blockages. Non-limiting examples ofantithrombotic and/or fibrinolytic agents include anticoagulants,anticoagulant antagonists, antiplatelet agents, thrombolytic agents,thrombolytic agent antagonists or combinations thereof. In certainembodiments, antithrombotic agents that can be administered orally, suchas, for example, aspirin and wafarin (coumadin), are preferred.

Non-limiting examples of anticoagulants include acenocoumarol, ancrod,anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextransulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate,ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium,oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin,picotamide, tioclomarol and warfarin.

Non-limiting examples of antiplatelet agents include aspirin, a dextran,dipyridamole (persantin), heparin, sulfinpyranone (anturane) andticlopidine (ticlid).

Non-limiting examples of thrombolytic agents include tissue plaminogenactivator (activase), plasmin, pro-urokinase, urokinase (abbokinase)streptokinase (streptase), anistreplase/APSAC (eminase).

In certain embodiments wherein a patient is suffering from a hemorrhageor an increased likelihood of hemorrhaging, an agent that may enhanceblood coagulation may be used. Non-limiting examples of bloodcoagulation promoting agents include thrombolytic agent antagonists andanticoagulant antagonists. Non-limiting examples of anticoagulantantagonists include protamine and vitamine K1.

Non-limiting examples of thrombolytic agent antagonists includeamiocaproic acid (amicar) and tranexamic acid (amstat). Non-limitingexamples of antithrombotics include anagrelide, argatroban, cilstazol,daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan,ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

Non-limiting examples of antiarrhythmic agents include Class Iantiarrhythmic agents (sodium channel blockers), Class II antiarrhythmicagents (beta-adrenergic blockers), Class III antiarrhythmic agents(repolarization prolonging drugs), Class IV antiarrhythmic agents(calcium channel blockers) and miscellaneous antiarrhythmic agents.

Non-limiting examples of sodium channel blockers include Class IA, ClassIB and Class IC antiarrhythmic agents. Non-limiting examples of Class IAantiarrhythmic agents include disppyramide (norpace), procainamide(pronestyl) and quinidine (quinidex). Non-limiting examples of Class IBantiarrhythmic agents include lidocaine (xylocalne), tocamide (tonocard)and mexiletine (mexitil). Non-limiting examples of Class ICantiarrhythmic agents include encamide (enkaid) and flecamide(tambocor).

Non-limiting examples of a beta blocker, otherwise known as aβ-adrenergic blocker, a β-adrenergic antagonist or a Class IIantiarrhythmic agent, include acebutolol (sectral), alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol,bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol,nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol,propanolol (inderal), sotalol (betapace), sulfinalol, talinolol,tertatolol, timolol, toliprolol and xibinolol. In certain embodiments,the beta blocker comprises an aryloxypropanolamine derivative.Non-limiting examples of aryloxypropanolamine derivatives includeacebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol,bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol,penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol andtoliprolol.

Non-limiting examples of an agent that prolong repolarization, alsoknown as a Class III antiarrhythmic agent, include amiodarone(cordarone) and sotalol (betapace).

Non-limiting examples of a calcium channel blocker, otherwise known as aClass IV antiarrythmic agent, include an arylalkylamine (e.g.,bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline,verapamil), a dihydropyridine derivative (felodipine, isradipine,nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) apiperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) ora micellaneous calcium channel blocker such as bencyclane, etafenone,magnesium, mibefradil or perhexyline. In certain embodiments a calciumchannel blocker comprises a long-acting dihydropyridine(nifedipine-type) calcium antagonist.

Non-limiting examples of miscellaneous antiarrhythmic agents includeadenosine (adenocard), digoxin (lanoxin), acecamide, ajmaline,amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine,capobenic acid, cifenline, disopyranide, hydroquinidine, indecamide,ipatropium bromide, lidocaine, lorajmine, lorcamide, meobentine,moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidinepolygalacturonate, quinidine sulfate and viquidil.

Non-limiting examples of antihypertensive agents include sympatholytic,alpha/beta blockers, alpha blockers, anti-angiotensin II agents, betablockers, calcium channel blockers, vasodilators and miscellaneousantihypertensives.

Non-limiting examples of an alpha blocker, also known as an α-adrenergicblocker or an α-adrenergic antagonist, include amosulalol, arotinolol,dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin,labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin andyohimbine. In certain embodiments, an alpha blocker may comprise aquinazoline derivative. Non-limiting examples of quinazoline derivativesinclude alfuzosin, bunazosin, doxazosin, prazosin, terazosin andtrimazosin. In certain embodiments, an antihypertensive agent is both analpha and beta adrenergic antagonist. Non-limiting examples of analpha/beta blocker comprise labetalol (normodyne, trandate).

Non-limiting examples of anti-angiotensin II agents include angiotensinconverting enzyme inhibitors and angiotensin II receptor antagonists.Non-limiting examples of angiotensin converting enzyme inhibitors (ACEinhibitors) include alacepril, enalapril (vasotec), captopril,cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril,perindopril, quinapril and ramipril. Non-limiting examples of anangiotensin II receptor blocker, also known as an angiotensin IIreceptor antagonist, an ANG receptor blocker or an ANG-II type-1receptor blocker (ARBS), include angiocandesartan, eprosartan,irbesartan, losartan and valsartan.

Non-limiting examples of a sympatholytic include a centrally actingsympatholytic or a peripherially acting sympatholytic. Non-limitingexamples of a centrally acting sympatholytic, also known as an centralnervous system (CNS) sympatholytic, include clonidine (catapres),guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).Non-limiting examples of a peripherally acting sympatholytic include aganglion blocking agent, an adrenergic neuron blocking agent, aβ-adrenergic blocking agent or a alpha1-adrenergic blocking agent.Non-limiting examples of a ganglion blocking agent include mecamylamine(inversine) and trimethaphan (arfonad). Non-limiting examples of anadrenergic neuron blocking agent include guanethidine (ismelin) andreserpine (serpasil). Non-limiting examples of a β-adrenergic blockerinclude acenitolol (sectral), atenolol (tenormin), betaxolol (kerlone),carteolol (cartrol), labetalol (normodyne, trandate), metoprolol(lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken),propranolol (inderal) and timolol (blocadren). Non-limiting examples ofalpha1-adrenergic blocker include prazosin (minipress), doxazocin(cardura) and terazosin (hytrin).

In certain embodiments a cardiovasculator therapeutic agent may comprisea vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or aperipheral vasodilator). In certain preferred embodiments, a vasodilatorcomprises a coronary vasodilator. Non-limiting examples of a coronaryvasodilator include amotriphene, bendazol, benfurodil hemisuccinate,benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep,dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane,etafenone, fendiline, floredil, ganglefene, herestrolbis(β-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin,lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin,pentaerythritol tetranitrate, pentrinitrol, perhexyline, pimethylline,trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine.

In certain embodiments, a vasodilator may comprise a chronic therapyvasodilator or a hypertensive emergency vasodilator. Non-limitingexamples of a chronic therapy vasodilator include hydralazine(apresoline) and minoxidil (loniten). Non-limiting examples of ahypertensive emergency vasodilator include nitroprusside (nipride),diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten)and verapamil.

Non-limiting examples of miscellaneous antihypertensives includeajmaline, γ-aminobutyric acid, bufeniode, cicletainine, ciclosidomine, acryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate,mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone,muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, aprotoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodiumnitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase andurapidil.

In certain embodiments, an antihypertensive may comprise anarylethanolamine derivative, a benzothiadiazine derivative, aN-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative,a guanidine derivative, a hydrazines/phthalazine, an imidazolederivative, a quanternary ammonium compound, a reserpine derivative or asuflonamide derivative. Non-limiting examples of arylethanolaminederivatives include amosulalol, bufuralol, dilevalol, labetalol,pronethalol, sotalol and sulfinalol. Non-limiting examples ofbenzothiadiazine derivatives include althizide, bendroflumethiazide,benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide,chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide,ethiazide, fenquizone, hydrochlorothizide, hydroflumethizide,methyclothiazide, meticrane, metolazone, paraflutizide, polythizide,tetrachlormethiazide and trichlormethiazide. Non-limiting examples ofN-carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril,cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril,moveltipril, perindopril, quinapril and ramipril. Non-limiting examplesof dihydropyridine derivatives include amlodipine, felodipine,isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine andnitrendipine. Non-limiting examples of guanidine derivatives includebethanidine, debrisoquin, guanabenz, guanacline, guanadrel, guanazodine,guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan.Non-limiting examples of hydrazines/phthalazines include budralazine,cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine,pheniprazine, pildralazine and todralazine. Non-limiting examples ofimidazole derivatives include clonidine, lofexidine, phentolamine,tiamenidine and tolonidine. Non-limiting examples of quanternaryammonium compounds include azamethonium bromide, chlorisondaminechloride, hexamethonium, pentacynium bis(methylsulfate), pentamethoniumbromide, pentolinium tartrate, phenactropinium chloride andtrimethidinium methosulfate. Non-limiting examples of reserpinederivatives include bietaserpine, deserpidine, rescinnamine, reserpineand syrosingopine. Non-limiting examples of sulfonamide derivativesinclude ambuside, clopamide, furosemide, indapamide, quinethazone,tripamide and xipamide.

Vasopressors generally are used to increase blood pressure during shock,which may occur during a surgical procedure. Non-limiting examples of avasopressor, also known as an antihypotensive, include amezinium methylsulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin,gepefrine, metaraminol, midodrine, norepinephrine, pholedrine andsynephrine.

Non-limiting examples of agents for the treatment of congestive heartfailure include anti-angiotensin II agents, afterload-preload reductiontreatment, diuretics and inotropic agents.

In certain embodiments, an animal patient that can not tolerate anangiotensin antagonist may be treated with a combination therapy. Suchtherapy may combine administration of hydralazine (apresoline) andisosorbide dinitrate (isordil, sorbitrate).

Non-limiting examples of a diuretic include a thiazide orbenzothiadiazine derivative (e.g., althiazide, bendroflumethazide,benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide,chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide,ethiazide, ethiazide, fenquizone, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, meticrane, metolazone,paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide),an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide,mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurouschloride, mersalyl), a pteridine (e.g., furtherene, triamterene),purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom,protheobromine, theobromine), steroids including aldosterone antagonists(e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative(e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone,diphenylmethane-4,4′-disulfonamide, disulfamide, ethoxzolamide,furosemide, indapamide, mefruside, methazolamide, piretanide,quinethazone, torasemide, tripamide, xipamide), a uracil (e.g.,aminometradine, amisometradine), a potassium sparing antagonist (e.g.,amiloride, triamterene) or a miscellaneous diuretic such as aminozine,arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine,isosorbide, mannitol, metochalcone, muzolimine, perhexyline, ticrnafenand urea.

Non-limiting examples of a positive inotropic agent, also known as acardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline,aminone, benfurodil hemisuccinate, bucladesine, cerberosine,camphotamide, convallatoxin, cymarin, denopamine, deslanoside,digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine,dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin,glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside,metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine,prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin,strphanthin, sulmazole, theobromine and xamoterol.

In particular embodiments, an intropic agent is a cardiac glycoside, abeta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limitingexamples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin(crystodigin). Non-limiting examples of a β-adrenergic agonist includealbuterol, bambuterol, bitolterol, carbuterol, clenbuterol,clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex),dopamine (intropin), dopexamine, ephedrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol,ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol andxamoterol. Non-limiting examples of a phosphodiesterase inhibitorinclude aminone (inocor).

Antianginal agents may comprise organonitrates, calcium channelblockers, beta blockers and combinations thereof.

Non-limiting examples of organonitrates, also known asnitrovasodilators, include nitroglycerin (nitro-bid, nitrostat),isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol,vaporole).

Endothelin (ET) is a 21-amino acid peptide that has potent physiologicand pathophysiologic effects that appear to be involved in thedevelopment of heart failure. The effects of ET are mediated throughinteraction with two classes of cell surface receptors. The type Areceptor (ET-A) is associated with vasoconstriction and cell growthwhile the type B receptor (ET-B) is associated with endothelial-cellmediated vasodilation and with the release of other neurohormones, suchas aldosterone. Pharmacologic agents that can inhibit either theproduction of ET or its ability to stimulate relevant cells are known inthe art. Inhibiting the production of ET involves the use of agents thatblock an enzyme termed endothelin-converting enzyme that is involved inthe processing of the active peptide from its precursor. Inhibiting theability of ET to stimulate cells involves the use of agents that blockthe interaction of ET with its receptors. Non-limiting examples ofendothelin receptor antagonists (ERA) include Bosentan, Enrasentan,Ambrisentan, Darusentan, Tezosentan, Atrasentan, Avosentan, Clazosentan,Edonentan, sitaxsentan, TBC 3711, BQ 123, and BQ 788.

In certain embodiments, the secondary therapeutic agent may comprise asurgery of some type, which includes, for example, preventative,diagnostic or staging, curative and palliative surgery. Surgery, and inparticular a curative surgery, may be used in conjunction with othertherapies, such as the present invention and one or more other agents.

Such surgical therapeutic agents for vascular and cardiovasculardiseases and disorders are well known to those of skill in the art, andmay comprise, but are not limited to, performing surgery on an organism,providing a cardiovascular mechanical prostheses, angioplasty, coronaryartery reperfusion, catheter ablation, providing an implantablecardioverter defibrillator to the subject, mechanical circulatorysupport or a combination thereof. Non-limiting examples of a mechanicalcirculatory support that may be used in the present invention comprisean intra-aortic balloon counterpulsation, left ventricular assist deviceor combination thereof.

In another embodiment, the present invention provides a method oftreating or preventing a musculoskeletal disorder in a subject in needthereof comprising (a) identifying a subject having or at risk of amusculoskeletal disorder; and (b) increasing the expression and/oractivity of one or more miR-15 family members in skeletal muscle cellsof said subject. The disorder may be selected from the group consistingof muscular dystrophy, disuse atrophy, muscle wasting in response toanti-gravity and denervation. Increasing the expression and/or activitymay comprise administering said one or more miR-15 family members tosaid subject, optionally comprised within a lipid vehicle, or maycomprise administering an expression vector that expresses said one ormore miR-15 family members in said subject. The expression vector may bea viral expression vector, such as an adenoviral expression vector, or anon-viral expression vector, such as one comprised within a lipidvehicle. The method may further comprise a non-miR-15 family membertherapy (i.e. another microRNA or other appropriate therapy).

The present invention also encompasses a pharmaceutical compositioncomprising an inhibitor of one or more miR-15 family members (e.g.miR-195, miR-497, miR-424, miR-15a, miR-15b, miR-16-1, and miR-16-2).The pharmaceutical composition may comprise any inhibitor of a miR-15family member as described herein, such as an antagomir, an antisenseoligonucleotide, an inhibitory RNA molecule, and a nucleic acidcomprising one or more miR-15 binding sites. Where clinical applicationsare contemplated, pharmaceutical compositions will be prepared in a formappropriate for the intended application. Generally, this will entailpreparing compositions that are essentially free of pyrogens, as well asother impurities that could be harmful to humans or animals.

Colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes, may beused as delivery vehicles for the oligonucleotide inhibitors of microRNAfunction or constructs expressing inhibitory nucleotides. Commerciallyavailable fat emulsions that are suitable for delivering the nucleicacids of the invention to tissues, such as cardiac muscle tissue,include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III, Nutrilipid,and other similar lipid emulsions. A preferred colloidal system for useas a delivery vehicle in vivo is a liposome (i.e., an artificialmembrane vesicle). The preparation and use of such systems is well knownin the art. Exemplary formulations are also disclosed in U.S. Pat. No.5,981,505; U.S. Pat. No. 6,217,900; U.S. Pat. No. 6,383,512; U.S. Pat.No. 5,783,565; U.S. Pat. No. 7,202,227; U.S. Pat. No. 6,379,965; U.S.Pat. No. 6,127,170; U.S. Pat. No. 5,837,533; U.S. Pat. No. 6,747,014;and WO03/093449, which are herein incorporated by reference in theirentireties.

One will generally desire to employ appropriate salts and buffers torender delivery vehicles stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the delivery vehicle or cells, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein, “pharmaceutically acceptablecarrier” includes solvents, buffers, solutions, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like acceptable for use in formulatingpharmaceuticals, such as pharmaceuticals suitable for administration tohumans. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredients of thepresent invention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions, provided they do not inactivate the vectors or cells ofthe compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection,or by direct injection into cardiac tissue. Pharmaceutical compositionscomprising miRNA inhibitors or expression constructs encoding inhibitorypolynucleotides may also be administered by catheter systems or systemsthat isolate coronary circulation for delivering therapeutic agents tothe heart. Various catheter systems for delivering therapeutic agents tothe heart and coronary vasculature are known in the art. Somenon-limiting examples of catheter-based delivery methods or coronaryisolation methods suitable for use in the present invention aredisclosed in U.S. Pat. No. 6,416,510; U.S. Pat. No. 6,716,196; U.S. Pat.No. 6,953,466, WO 2005/082440, WO 2006/089340, U.S. Patent PublicationNo. 2007/0203445, U.S. Patent Publication No. 2006/0148742, and U.S.Patent Publication No. 2007/0060907, which are all herein incorporatedby reference in their entireties. Such compositions would normally beadministered as pharmaceutically acceptable compositions, as describedsupra.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use or catheterdelivery include, for example, sterile aqueous solutions or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersions. Generally, these preparations aresterile and fluid to the extent that easy injectability exists.Preparations should be stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Appropriate solvents ordispersion media may contain, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, an inhibitor of one or more miR-15 family members,such as an antagomir, is included in a kit. The kit may contain two ormore, three or more, four or more, five or more, or six inhibitors foreach miR-15 family member. By way of example, the kit may contain aninhibitor of miR-195 and an inhibitor of miR-15a. All possiblecombinations of inhibitors for miR-15 family members are contemplated bythe invention. The kit may further include water and/or buffers tostabilize the inhibitory polynucleotides. The kit may also include oneor more transfection reagent(s) to facilitate delivery of the miRNAinhibitors to cells.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit (labelingreagent and label may be packaged together), the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a vial. The kits of thepresent invention also will typically include a means for containing thenucleic acids, and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which thenucleic acid formulations are placed, preferably, suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

Such kits may also include components that preserve or maintain themiRNA inhibitors or that protect against their degradation. Suchcomponents may be RNAse-free or protect against RNAses. Such kitsgenerally will comprise, in suitable means, distinct containers for eachindividual reagent or solution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented. A kit may also includeutensils or devices for administering the miRNA inhibitor by variousadministration routes, such as parenteral or catheter administration.

It is contemplated that such reagents are embodiments of kits of theinvention. Such kits, however, are not limited to the particular itemsidentified above and may include any reagent used for the manipulationor characterization of miRNA.

The present invention further comprises methods for identifyingmodulators of miR-15 family members. Identified inhibitors of thefunction of one or more miR-15 family members are useful in theprevention or treatment or reversal of cardiac hypertrophy or heartfailure. Modulators (e.g. inhibitors) of miR-15 family members may beincluded in pharmaceutical compositions for the treatment of cardiacdisorders according to the methods of the present invention.

These assays may comprise random screening of large libraries ofcandidate compounds; alternatively, the assays may be used to focus onparticular classes of compounds selected with an eye towards structuralattributes that are believed to make them more likely to inhibit theexpression and/or function of miR-15 family members.

To identify a modulator of a miR-15 family member, one generally willdetermine the function of a miR-15 family member in the presence andabsence of the candidate compound. For example, a method generallycomprises:

-   -   (a) providing a candidate compound;    -   (b) admixing the candidate compound with a miR-15 family member;    -   (c) measuring activity of the miR-15 family member; and    -   (d) comparing the activity in step (c) with the activity in the        absence of the candidate compound,

wherein a difference between the measured activities indicates that thecandidate compound is a modulator of a miR-15 family member.

Assays also may be conducted in isolated cells, organs, or in livingorganisms.

Assessing the activity or expression of a miR-15 family member maycomprise assessing the expression level of the miR-15 family member.Those in the art will be familiar with a variety of methods forassessing RNA expression levels including, for example, northernblotting or RT-PCR. Assessing the activity or expression of the miR-15family member may comprise assessing the activity of the miR-15 familymember. In some embodiments, assessing the activity of the miR-15 familymember comprises assessing expression or activity of a gene regulated bythe miR-15 family member. Genes regulated by miR-15 family membersinclude, for example, FGF2, TGFb-induced factor 2, BCL9I, BCL2L, CDC25A,cyclin E1, cyclin D1, and cyclin D2. Those in the art will be familiarwith a variety of methods for assessing the activity or expression ofgenes regulated by miR-15 family members. Such methods include, forexample, northern blotting, RT-PCR, ELISA, or western blotting.

It will, of course, be understood that all the screening methods of thepresent invention are useful in themselves notwithstanding the fact thateffective candidates may not be found. The invention provides methodsfor screening for such candidates, not solely methods of finding them.

As used herein the term “candidate substance” refers to any moleculethat may potentially modulate the function of miR-15 family members. Onewill typically acquire, from various commercial sources, molecularlibraries that are believed to meet the basic criteria for useful drugsin an effort to “brute force” the identification of useful compounds.Screening of such libraries, including combinatorially-generatedlibraries (e.g., antagomir libraries), is a rapid and efficient way toscreen large number of related (and unrelated) compounds for activity.Combinatorial approaches also lend themselves to rapid evolution ofpotential drugs by the creation of second, third, and fourth generationcompounds modeled on active, but otherwise undesirable compounds.Non-limiting examples of candidate compounds that may be screenedaccording to the methods of the present invention are proteins,peptides, polypeptides, polynucleotides, oligonucleotides or smallmolecules. Modulators of miR-15 family members may also be agonists orinhibitors of upstream regulators of any one of the miR-15 familymembers.

A quick, inexpensive and easy assay to run is an in vitro assay. Suchassays generally use isolated molecules, can be run quickly and in largenumbers, thereby increasing the amount of information obtainable in ashort period of time. A variety of vessels may be used to run theassays, including test tubes, plates, dishes and other surfaces such asdipsticks or beads.

A technique for high throughput screening of compounds is described inWO 84/03564, which is herein incorporated by reference in its entirety.Large numbers of small antagomir compounds may be synthesized on a solidsubstrate, such as plastic pins or some other surface. Such moleculescan be rapidly screening for their ability to hybridize to miR-15 familymembers.

The present invention also contemplates the screening of compounds fortheir ability to modulate expression and/or function of one or moremiR-15 family members in cells. Various cell lines, including thosederived from skeletal muscle cells, can be utilized for such screeningassays, including cells specifically engineered for this purpose.Primary cardiac cells also may be used, as can the H9C2 cell line.

In vivo assays involve the use of various animal models of heartdisease, including transgenic animals, that have been engineered to havespecific defects, or carry markers that can be used to measure theability of a candidate compound to reach and affect different cellswithin the organism. Due to their size, ease of handling, andinformation on their physiology and genetic make-up, mice are apreferred embodiment, especially for transgenics. However, other animalsare suitable as well, including rats, rabbits, hamsters, guinea pigs,gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses andmonkeys (including chimps, gibbons and baboons). Assays for inhibitorsmay be conducted using an animal model derived from any of thesespecies.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route that could be utilized for clinical purposes.Determining the effectiveness of a compound in vivo may involve avariety of different criteria, including but not limited to alterationof hypertrophic signaling pathways and physical symptoms of hypertrophy.Also, measuring toxicity and dose response can be performed in animalsin a more meaningful fashion than in in vitro or in cyto assays.

In one embodiment, the present invention provides a method of regulatingcardiac cell survival comprising administering to cardiac cells amodulator of one or more miR-15 family members. In another embodiment,the modulator is an agonist of the expression or activity of a miR-15family member. In another embodiment, cardiac cell survival is decreasedfollowing administration of an agonist of a miR-15 family member. Inanother embodiment, the modulator of a miR-15 family member is aninhibitor of the expression or activity of a miR-15 family member. Instill another embodiment, cardiac cell survival is increased followingadministration of an inhibitor of a miR-15 family member.

In a further embodiment, the present invention provides a method ofregulating apoptosis of cardiac cells comprising administering tocardiac cells a modulator of one or more miR-15 family members. Inanother embodiment, the modulator is an agonist of the expression oractivity of a miR-15 family member. In another embodiment, apoptosis ofcardiac cells is increased following administration of an agonist of amiR-15 family member. In another embodiment, the modulator of a miR-15family member is an inhibitor of the expression or activity of a miR-15family member. In still another embodiment, apoptosis of cardiac cellsis decreased following administration of an inhibitor of a miR-15 familymember. In some embodiments, the expression of FGF2, TGFb-induced factor2, BCL9I, BCL2L, CDC25A, cyclin E1, cyclin D1, or cyclin D2 is increasedin a cell by contacting the cell with a miR-15 family inhibitor. Inother embodiments, expression of FGF2, TGFb-induced factor 2, BCL9I,BCL2L, CDC25A, cyclin E1, cyclin D1, or cyclin D2 is decreased in a cellby contacting the cell with a miR-15 family agonist.

Thus, the present invention includes a method of regulating expressionof FGF2, TGFb-induced factor 2, BCL9I, BCL2L, CDC25A, cyclin E1, cyclinD1, or cyclin D2 in a cell comprising contacting the cell with amodulator of a miR-15 family member. In one embodiment, the expressionof FGF2, TGFb-induced factor 2, BCL9I, BCL2L, CDC25A, cyclin E1, cyclinD1, or cyclin D2 is decreased in the cell following administration of amiR-15 family agonist. In another embodiment, the expression of FGF2,TGFb-induced factor 2, BCL9I, BCL2L, CDC25A, cyclin E1, cyclin D1, orcyclin D2 is increased in the cell following administration of a miR-15family inhibitor.

An agonist of a miR-15 family member may be a polynucleotide comprisinga mature sequence or a star sequence from a miR-15 family member. In oneembodiment, the polynucleotide comprises the sequence of SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 20, or SEQ ID NO: 21. In another embodiment, the agonistof a miR-15 family member may be a polynucleotide comprising thepri-miRNA or pre-miRNA sequence for a miR-15 family member (e.g.pre-miR-195, pre-miR-497, pre-miR-424, pre-miR-15a, pre-miR-15b,pre-miR-16-1, or pre-miR-16-2). For example, in one embodiment, theagonist may be a polynucleotide comprising a sequence of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, orSEQ ID NO: 19. The polynucleotide comprising the a mature sequence of amiR-15 family member may be single stranded or double stranded. Thepolynucleotides may contain one or more chemical modifications, such aslocked nucleic acids, peptide nucleic acids, sugar modifications, suchas 2′-O-alkyl (e.g. 2′-O-methyl, 2′-O-methoxyethyl), 2′-fluoro, and 4′thio modifications, and backbone modifications, such as one or morephosphorothioate, morpholino, or phosphonocarboxylate linkages. In oneembodiment, the polynucleotide comprising a miR-15 family membersequence is conjugated to cholesterol. In another embodiment, theagonist of a miR-15 family member may be an agent distinct from themiR-15 family member that acts to increase, supplement, or replace thefunction of the miR-15 family member. In another embodiment, the miR-15family agonist may be expressed in vivo from a vector.

In one embodiment, the present invention provides a method for treatingpathologic cardiac hypertrophy, heart failure, or myocardial infarctionin a subject in need thereof comprising: identifying a subject havingcardiac hypertrophy, heart failure, or myocardial infarction; andadministering an inhibitor of one or more miR-15 family members to thesubject. In certain embodiments of the invention the miR-15 familyinhibitor may be identified by a method comprising: (a) contacting acell with a candidate compound; (b) assessing activity or expression ofa miR-15 family member; and (c) comparing the activity or expression instep (b) with the activity or expression in the absence of the candidatecompound, wherein a reduction in the activity or expression of themiR-15 family member in the cell contacted with the candidate compoundcompared to the activity or expression in the cell in the absence of thecandidate compound indicates that the candidate compound is an inhibitorof the miR-15 family member.

A particular embodiment of the present invention provides transgenicanimals that lack one or both functional alleles of a miR-15 familymember. Also, transgenic animals that express miR-15 family membersunder the control of an inducible, tissue selective or a constitutivepromoter, recombinant cell lines derived from such animals, andtransgenic embryos may be useful in determining the exact role that themiR-15 family member plays in the development and differentiation ofcardiomyocytes and in the development of pathologic cardiac hypertrophyand heart failure. Furthermore, these transgenic animals may provide aninsight into heart development. The use of constitutively expressedmiR-15 family members provides a model for over- or unregulatedexpression. Also, transgenic animals that are “knocked out” for one ormore miR-15 family members, in one or both alleles, are contemplated.

In a general embodiment, a transgenic animal is produced by theintegration of a given transgene into the genome in a manner thatpermits the expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; incorporated herein by reference), and Brinster etal. (1985; incorporated herein by reference).

Typically, a gene flanked by genomic sequences is transferred bymicroinjection into a fertilized egg. The microinjected eggs areimplanted into a host female, and the progeny are screened for theexpression of the transgene. Transgenic animals may be produced from thefertilized eggs from a number of animals including, but not limited toreptiles, amphibians, birds, mammals, and fish.

DNA clones for microinjection can be prepared by any means known in theart. For example, DNA clones for microinjection can be cleaved withenzymes appropriate for removing the bacterial plasmid sequences, andthe DNA fragments electrophoresed on 1% agarose gels in TBE buffer,using standard techniques. The DNA bands are visualized by staining withethidium bromide, and the band containing the expression sequences isexcised. The excised band is then placed in dialysis bags containing 0.3M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags,extracted with a 1:1 phenol:chloroform solution and precipitated by twovolumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer(0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on anElutip-D™ column. The column is first primed with 3 ml of high saltbuffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washingwith 5 ml of low salt buffer. The DNA solutions are passed through thecolumn three times to bind DNA to the column matrix. After one wash with3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt bufferand precipitated by two volumes of ethanol. DNA concentrations aremeasured by absorption at 260 nm in a UV spectrophotometer. Formicroinjection, DNA concentrations are adjusted to 3 μg/ml in 5 mM Tris,pH 7.4 and 0.1 mM EDTA. Other methods for purification of DNA formicroinjection are described in Palmiter et al. (1982); and in Sambrooket al. (2001).

In an exemplary microinjection procedure, female mice six weeks of ageare induced to superovulate with a 5 IU injection (0.1 cc, ip) ofpregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours laterby a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG;Sigma). Females are placed with males immediately after hCG injection.Twenty-one hours after hCG injection, the mated females are sacrificedby C02 asphyxiation or cervical dislocation and embryos are recoveredfrom excised oviducts and placed in Dulbecco's phosphate buffered salinewith 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cellsare removed with hyaluronidase (1 mg/ml). Pronuclear embryos are thenwashed and placed in Earle's balanced salt solution containing 0.5% BSA(EBSS) in a 37.5° C. incubator with a humidified atmosphere at 5% CO₂,95% air until the time of injection. Embryos can be implanted at thetwo-cell stage.

Randomly cycling adult female mice are paired with vasectomized males.C57BL/6 or Swiss mice or other comparable strains can be used for thispurpose. Recipient females are mated at the same time as donor females.At the time of embryo transfer, the recipient females are anesthetizedwith an intraperitoneal injection of 0.015 ml of 2.5% avertin per gramof body weight. The oviducts are exposed by a single midline dorsalincision. An incision is then made through the body wall directly overthe oviduct. The ovarian bursa is then torn with watchmakers forceps.Embryos to be transferred are placed in DPBS (Dulbecco's phosphatebuffered saline) and in the tip of a transfer pipet (about 10 to 12embryos). The pipet tip is inserted into the infundibulum and theembryos transferred. After the transfer, the incision is closed by twosutures.

As used herein, the term “heart failure” is broadly used to mean anycondition that reduces the ability of the heart to pump blood. As aresult, congestion and edema develop in the tissues. Most frequently,heart failure is caused by decreased contractility of the myocardium,resulting from reduced coronary blood flow; however, many other factorsmay result in heart failure, including damage to the heart valves,vitamin deficiency, and primary cardiac muscle disease. Though theprecise physiological mechanisms of heart failure are not entirelyunderstood, heart failure is generally believed to involve disorders inseveral cardiac autonomic properties, including sympathetic,parasympathetic, and baroreceptor responses. The phrase “manifestationsof heart failure” is used broadly to encompass all of the sequelaeassociated with heart failure, such as shortness of breath, pittingedema, an enlarged tender liver, engorged neck veins, pulmonary ralesand the like including laboratory findings associated with heartfailure.

The term “treatment” or grammatical equivalents encompasses theimprovement and/or reversal of the symptoms of heart failure (i.e., theability of the heart to pump blood). “Improvement in the physiologicfunction” of the heart may be assessed using any of the measurementsdescribed herein (e.g., measurement of ejection fraction, fractionalshortening, left ventricular internal dimension, heart rate, etc.), aswell as any effect upon the animal's survival. In use of animal models,the response of treated transgenic animals and untreated transgenicanimals is compared using any of the assays described herein (inaddition, treated and untreated non-transgenic animals may be includedas controls). A compound which causes an improvement in any parameterassociated with heart failure used in the screening methods of theinstant invention may thereby be identified as a therapeutic compound.

The term “dilated cardiomyopathy” refers to a type of heart failurecharacterized by the presence of a symmetrically dilated left ventriclewith poor systolic contractile function and, in addition, frequentlyinvolves the right ventricle.

The term “compound” refers to any chemical entity, pharmaceutical, drug,and the like that can be used to treat or prevent a disease, illness,sickness, or disorder of bodily function. Compounds comprise both knownand potential therapeutic compounds. A compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. A “known therapeutic compound” refers to a therapeuticcompound that has been shown (e.g., through animal trials or priorexperience with administration to humans) to be effective in suchtreatment. In other words, a known therapeutic compound is not limitedto a compound efficacious in the treatment of heart failure.

As used herein, the term “cardiac hypertrophy” refers to the process inwhich adult cardiac myocytes respond to stress through hypertrophicgrowth. Such growth is characterized by cell size increases without celldivision, assembling of additional sarcomeres within the cell tomaximize force generation, and an activation of a fetal cardiac geneprogram. Cardiac hypertrophy is often associated with increased risk ofmorbidity and mortality, and thus studies aimed at understanding themolecular mechanisms of cardiac hypertrophy could have a significantimpact on human health.

As used herein, the term “modulate” refers to a change or an alterationin a biological activity. Modulation may be an increase or a decrease inprotein activity, a change in kinase activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties associated with the activity of a protein orother structure of interest. The term “modulator” refers to any moleculeor compound which is capable of changing or altering biological activityas described above.

The term “β-adrenergic receptor antagonist” refers to a chemicalcompound or entity that is capable of blocking, either partially orcompletely, the beta (β) type of adrenoreceptors (i.e., receptors of theadrenergic system that respond to catecholamines, especiallynorepinephrine). Some β-adrenergic receptor antagonists exhibit a degreeof specificity for one receptor subtype (generally β₁); such antagonistsare termed “β₁-specific adrenergic receptor antagonists” and“β₂-specific adrenergic receptor antagonists.” The term β-adrenergicreceptor antagonist” refers to chemical compounds that are selective andnon-selective antagonists. Examples of β-adrenergic receptor antagonistsinclude, but are not limited to, acebutolol, atenolol, butoxamine,carteolol, esmolol, labetolol, metoprolol, nadolol, penbutolol,propanolol, and timolol. The use of derivatives of known β-adrenergicreceptor antagonists is encompassed by the methods of the presentinvention. Indeed any compound, which functionally behaves as aβ-adrenergic receptor antagonist is encompassed by the methods of thepresent invention.

The terms “angiotensin-converting enzyme inhibitor” or “ACE inhibitor”refer to a chemical compound or entity that is capable of inhibiting,either partially or completely, the enzyme involved in the conversion ofthe relatively inactive angiotensin I to the active angiotensin II inthe rennin-angiotensin system. In addition, the ACE inhibitorsconcomitantly inhibit the degradation of bradykinin, which likelysignificantly enhances the antihypertensive effect of the ACEinhibitors. Examples of ACE inhibitors include, but are not limited to,benazepril, captopril, enalopril, fosinopril, lisinopril, quiapril andramipril. The use of derivatives of known ACE inhibitors is encompassedby the methods of the present invention. Indeed any compound, whichfunctionally behaves as an ACE inhibitor, is encompassed by the methodsof the present invention.

As used herein, the term “genotypes” refers to the actual geneticmake-up of an organism, while “phenotype” refers to physical traitsdisplayed by an individual. In addition, the “phenotype” is the resultof selective expression of the genome (i.e., it is an expression of thecell history and its response to the extracellular environment). Indeed,the human genome contains an estimated 30,000-35,000 genes. In each celltype, only a small (i.e., 10-15%) fraction of these genes are expressed.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Furthermore, compositions and kits of the invention canbe used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The following examples are included to further illustrate variousaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques and/or compositions discovered by the inventors tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1 Regulation of Cardiac Hypertrophy and Heart Failureby Stress-Responsive miRNAs

In light of their involvement in modulating cellular phenotypes, theinventors have hypothesized that miRNAs may play a role in regulatingthe response of the heart to cardiac stress, which is known to result intranscriptional and translational changes in gene expression. Toinvestigate the potential involvement of miRNAs in cardiac hypertrophy,a side-by-side miRNA microarray analysis was performed in twoestablished mouse models of cardiac hypertrophy, using a microarray thatrepresented 186 different miRNAs. Mice that were subjected to thoracicaortic banding (TAB), which induces hypertrophy by increased afterloadon the heart (Hill et al., 2000), were compared to sham operatedanimals. In a second model, transgenic mice expressing activatedcalcineurin (CnA) in the heart, which results in a severe,well-characterized form of hypertrophy (Molkentin et al., 1998), werecompared to wild-type littermates (FIG. 1A). RNA isolated from hearts ofmice subjected to TAB showed increased expression of 27 miRNAs comparedto sham-operated controls, and CnA Tg mice showed increased expressionof 33 miRNAs compared with non-transgenic littermate controls, of which21 were up-regulated in both models. Similarly, TAB and CnA-inducedhypertrophy were accompanied by reduced expression of 15 and 14 miRNAs,respectively, of which 7 miRNAs were down-regulated in common (FIG. 1B).Northern analysis of expression of these miRNAs and previous microarrayanalyses (Barad et al., 2004; Sempere et al., 2004; Shingara et al.,2005; Babak et al., 2004; Liu et al., 2004) indicate that they areexpressed in a wide range of tissues.

Based on their relative expression levels, conservation of human, ratand mouse sequences, and levels of expression during hypertrophy, theinventors focused on 11 up- and 5 down-regulated miRNAs. Northern blotanalysis of RNA isolated from cardiac tissue from WT and CnA Tg animalsconfirmed an increased expression of miR-21, miR-23, miR-24, miR-125b,miR-195, miR-199a, and miR-214, and decreased expression of miR-29c,miR-93, miR-150 and miR-181b (FIG. 1C). Collectively, these dataindicate that distinct miRNAs are regulated during cardiac hypertrophy,suggesting the possibility that they might function as modulators ofthis process.

Ventricular hypertrophy develops in response to numerous forms ofcardiac stress and often leads to heart failure in humans (Arad et al.,2002). Northern blot analysis of the hypertrophy-regulated miRNAs inidiopathic end-stage failing human hearts showed increased expression ofmiR-24, miR-125b, miR-195, miR-199a and miR-214, while the expressionfor miR-23 appeared to be variable within the non-failing and failinggroups (FIG. 2). No change in expression of miR-21, miR-27, miR-29c,miR-93, miR-150 and miR-181b was found (data not shown). Thus, thealtered pattern of miRNA expression in the failing human heartoverlapped with that of the hypertrophic mouse heart, suggesting thatthese miRNAs represent a molecular signature of adverse cardiacremodeling.

Example 2 Cardiac Over-Expression of miR-195 is Sufficient to DriveCardiac Hypertrophy

MiR-24, miR-195 and miR-214 were overexpressed specifically in the heartunder the control of the α-myosin heavy chain (MHC) promoter. F1offspring could not be obtained for miR-24, suggesting that cardiacover-expression of this miRNA causes embryonic lethality. Since alloffspring of the miR-195 transgenic (Tg) line 3 died in the first twoweeks after birth due to heart failure (FIG. 3), the Tg line 1 formiR-195, which was viable, was used for further studies. Northern blotanalysis showed miR-195 to be expressed at levels ˜25-fold above normalin Tg line 1 (FIG. 3). Over-expression of miR-195 initially inducedcardiac growth with disorganization of cardiomyocytes, which progressedto a dilated phenotype by 6 weeks of age. Although there were somefibrotic lesions, the dramatic increase in size of individual myocytesin miR-195 Tg mice compared to wild-type (WT) was more striking (FIG.3).

Echocardiography on 6 week-old animals showed that miR-195 Tg micedisplayed thinning of the left ventricular walls (AWs and PWs), anincrease in left ventricular diameter (LVIDd and LVIDs) and adeterioration in cardiac function, as indicated by decreased fractionalshortening (FIG. 4A). Heart weight to body weight ratios were alsodramatically increased in miR-195 Tg animals compared to WT littermates,indicating that over-expression of miR-195 was sufficient to stimulatecardiac growth (FIG. 4B). Real-time PCR analysis on cardiac tissue frommiR-195 Tg animals compared to their WT littermates, revealed dramaticup-regulation of the hypertrophic markers atrial natriuretic factor(ANF), b-type natriuretic protein (BNP) and β-myosin heavy chain (βMHC)in response to cardiac over-expression of miR-195 (FIG. 4C).

In contrast to the dramatic effects of overexpression of miR-195 oncardiac structure, function, and gene expression, cardiacover-expression of miR-214 at levels comparable to those of miR-195 hadno phenotypic effect (FIGS. 4C and 5, and data not shown). Thus, thecardiac remodeling induced in the miR-195 Tg animals is specifically dueto functional effects of this miRNA rather than a general non-specificeffect resulting from miRNA over-expression. These results indicate thatincreased expression of miR-195 induces hypertrophic signaling leadingto cardiac failure. Since miR-195 belongs to a small family of relatedmiRNAs, the miR-15 family, other family members are also likely toparticipate in cardiac disease.

The ability of miR-195 to promote cardiac growth contrasts with that ofmiR-1, a muscle-specific miRNA that inhibits cardiac growth bysuppressing the expression of the bHLH protein Hand2 (Zhao et al.,2005). miR-1 is highly expressed in the adult heart, but miR-195 isapparently capable of over-riding the inhibitory influence of miR-1 oncardiac growth.

It is especially interesting that overexpression in cardiomyocytes ofthe miRNAs that were down-regulated during hypertrophy caused anapparent reduction in cell size, an effect opposite than that evoked bythe upregulated miRNAs. One interpretation of these results is thatthese miRNAs normally function to suppress growth and are thereforedown-regulated to enhance hypertrophy.

miRNA-195 belongs to a small family of microRNAs, the miR-15 family,which contains miR-195, miR-16-1, miR-15a, miR-15b, miR-16-2, miR-424,and miR-497. Four of the miR-15 family members are expressed as threeclustered transcripts (FIG. 6A). Using a variety of bioinformaticsapproaches, potential mRNA targets for miR-195 were identified. Severalof the identified target mRNAs encode proteins involved in cellproliferation, survival and anti-apoptosis (FIG. 6B). One of thepredicted targets for the miR-15 family is FGF2, which has been shown topromote cardiac repair (FIG. 7). All members of the miR-15 family areup-regulated in failing human hearts, indicating that this family ofmicroRNAs plays a key role in pathological cardiac remodeling (FIG. 8).Since all the miR-15 family members share a conserved seed sequence, allmiR-15 family members could be inhibited simultaneously by targeting theseed sequence. One such approach entails overexpression of a nucleicacid containing multiple binding sites, which comprise a sequencecomplementary to the seed sequence. The nucleic acid would “scavenge” or“sponge” all separate members because of their overlap in seed regionand consequently overlap in target sequence (FIG. 8B) (Ebert et al.,2007).

All publications, patents, and patent applications discussed and citedherein are incorporated herein by reference in their entireties. All ofthe compositions and methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods, and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

X. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of treating pathologic cardiachypertrophy, heart failure, or myocardial infarction in a subject inneed thereof comprising administering to said subject an inhibitor oftwo or more miR-15 family members, wherein the inhibitor is a nucleicacid comprising a sequence that is substantially complementary to thesequence of SEQ ID NO: 18, and wherein the expression or activity ofsaid two or more miR-15 family members is reduced in the heart cells ofthe subject following administration of the inhibitor.
 2. The method ofclaim 1, wherein the nucleic acid is an antisense oligonucleotide. 3.The method of claim 1, wherein said two or more miR-15 family membersare selected from the group consisting of miR-15a, miR-15b, miR-16,miR-195, miR-424, and miR-497.
 4. The method of claim 2, wherein theantisense oligonucleotide comprises a sequence that is at leastpartially complementary to a mature sequence of a miR-15 family member.5. The method of claim 4, wherein the antisense oligonucleotidecomprises a sequence that is at least partially complementary to asequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO:
 20. 6.The method of claim 1, wherein the inhibitor of two or more miR-15family members is administered by parenteral, oral, subcutaneous,intravenous, intramuscular, intradermal, intraperitoneal, transdermal,sustained release, controlled release, delayed release, suppository,catheter, or sublingual administration or direct injection into cardiactissue.
 7. The method of claim 1, further comprising administering tosaid subject a second therapy.
 8. The method of claim 7, wherein saidsecond therapy is selected from the group consisting of a beta blocker,an ionotrope, a diuretic, ACE-I, All antagonist, BNP, a Ca++-blocker, anendothelin receptor antagonist, and an HDAC inhibitor.
 9. The method ofclaim 1, wherein one or more symptoms of pathologic cardiac hypertrophy,heart failure, or myocardial infarction is improved in the subjectfollowing administration of the inhibitor of two or more miR-15 familymembers.
 10. The method of claim 2, wherein the antisenseoligonucleotide has at least one chemical modification.
 11. The methodof claim 10, wherein said chemical modification is a sugar modificationor a backbone modification.
 12. The method of claim 10, wherein saidchemical modification is a locked nucleic acid.
 13. The method of claim2, wherein said antisense oligonucleotide is about 19 to about 25nucleotides in length.
 14. The method of claim 2, wherein said antisenseoligonucleotide is about 15 nucleotides in length.
 15. The method ofclaim 9, wherein said improved symptoms include increased exercisecapacity, increased cardiac ejection volume, decreased left ventricularend diastolic pressure, decreased pulmonary capillary wedge pressure,increased cardiac output, increased cardiac index, lowered pulmonaryartery pressures, decreased left ventricular end systolic and diastolicdimensions, decreased left and right ventricular wall stress, decreasedwall tension, and reduction in infarct size.
 16. The method of claim 11,wherein the sugar modification is a 2′-O-alkyl, 2′-O-methyl,2′-O-methoxyethyl, 2′-fluoro, or 4′-thio modification.
 17. The method ofclaim 11, wherein the backbone modification is a phosphorothioatelinkage.
 18. The method of claim 1, wherein the nucleic acid consists ofa sequence that is complementary to the sequence of SEQ ID NO:
 18. 19.The method of claim 1, wherein the expression or activity of three ormore miR-15 family members is reduced in the heart cells of the subjectfollowing administration of the inhibitor.
 20. The method of claim 1,wherein the expression or activity of all miR-15 family members isreduced in the heart cells of the subject following administration ofthe inhibitor.
 21. The method of claim 1, wherein the nucleic acidcomprises a sequence that is fully complementary to the sequence of SEQID NO:
 18. 22. The method of claim 5, wherein the antisenseoligonucleotide comprises a sequence that is at least 85% complementaryto a sequence selected from the group consisting of: SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO:20.
 23. The method of claim 5, wherein the antisense oligonucleotidecomprises a sequence that is at least 95% complementary to a sequenceselected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 9, SEQID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO:
 20. 24. Themethod of claim 1, wherein the nucleic acid comprises two or morebinding sites, and wherein each binding site comprises a sequence thatis substantially complementary to the sequence of SEQ ID NO:
 18. 25. Themethod of claim 24, wherein each binding site comprises a sequence thatis fully complementary to the sequence of SEQ ID NO:
 18. 26. The methodof claim 24, wherein the nucleic acid is expressed from a vector underthe control of a cardiac-specific promoter.
 27. The method of claim 26,wherein the cardiac-specific promoter is an alpha myosin heavy chainpromoter.